Dux R, Kindler-Röhrborn A, Lennartz K, Rajewsky M F
Institute of Cell Biology (Cancer Research), West German Cancer Center Essen, University of Essen Medical School, F.R.G.
J Immunol Methods. 1991 Nov 22;144(2):175-83. doi: 10.1016/0022-1759(91)90084-s.
Monoclonal antibodies (Mabs) directed against cell surface determinants and conjugated to fluochromes, radionuclids or drugs are of increasing importance in cell and tumor biology as well as in clinical oncology. Many of the applications of Mab require precise and quantitative information regarding the molecular interactions of labeled antibody with the respective antigen expressed on the cell surface. These interactions are characterized by the association rate constant (ka), the dissociation rate constant (kd) and the antibody affinity constant (K). The immunoreactive fraction (IRF) of the labeled antibody molecules directly influences these parameters. IRF is usually reduced below 100% by antibody purification and labeling procedures and, in case of radiolabeled antibodies, by radiation damage during antibody storage. Besides the calculation of kinetic parameters, IRF should, therefore, be determined for the quality control of any antibody preparation before experimental or clinical application. Commonly used methods for measuring IRF are based on radioimmunoassays (RIA) on intact cells performed under antigen excess. However, especially with Mabs directed against cell surface antigens expressed in small numbers per cell and for displaying low affinity constants, these assays often give unsatisfactory results. We have, therefore, established a method which permits us to determine IRF, ka, kd and K for an 125I-labeled Mab with precision even in the absence of antigen excess.
针对细胞表面决定簇并与荧光染料、放射性核素或药物偶联的单克隆抗体（Mab）在细胞和肿瘤生物学以及临床肿瘤学中的重要性日益增加。Mab的许多应用需要关于标记抗体与细胞表面表达的相应抗原的分子相互作用的精确和定量信息。这些相互作用的特征在于结合速率常数（ka）、解离速率常数（kd）和抗体亲和常数（K）。标记抗体分子的免疫反应分数（IRF）直接影响这些参数。通过抗体纯化和标记程序，IRF通常会降低到100%以下，对于放射性标记抗体，在抗体储存期间还会受到辐射损伤。因此，除了计算动力学参数外，在实验或临床应用之前，应该测定IRF以控制任何抗体制剂的质量。常用的测量IRF的方法基于在抗原过量条件下对完整细胞进行的放射免疫分析（RIA）。然而，特别是对于针对每个细胞中少量表达且亲和力常数较低的细胞表面抗原的Mab，这些分析常常给出不令人满意的结果。因此，我们建立了一种方法，即使在没有抗原过量的情况下，也能精确地测定125I标记的Mab的IRF、ka、kd和K。
